In this study we explored peptide binding and neutralization assays and their relation to predicting disease progression and viral transmission. Biologically relevant polyclonal and monoclonal neutralizing antibodies that were derived from natural human immunodeficiency virus type 1 (HIV- 1) infection of humans, experimental infections of chimpanzees, and viral envelope protein peptide immunizations were characterized. Comparison of V3-specific monoclonal antibodies by antigen-limited enzyme linked- immunosorbent assay and a quantitative HIV-1 neutralization assay demonstrated a less than optimal predictive relationship between binding and neutralization potency. On the other hand, polyclonal sera from goats immunized with B3-specific peptides derived from three different HIV-1 strains, as well as sera from other HIV-1-infected individuals, demonstrated correlation between binding affinity and neutralization. A novel peptide immunogen walking strategy was used to map the neutralization activity of potential epitopes near the fusion segment at the amino terminus of gp41. The constructs were immunogenic, and the antibody elicited revealed two novel neutralizing epitopes in this relatively conserved and immunosilent region, a proximal MGA-QARQ site and a distal QLT site. These antibodies neutralized northern hemisphere, Caribbean, and African HIV-1 isolates. We are currently investigating their biological relevancy through kinetic neutralization and synergy analysis and their ability to block cell-to-cell fusion. Optimized immunogens from this region may be useful in the development of an effective vaccine for acquired immunodeficiency syndrome. During basic virologic studies of HIV-1, multiple physical-chemical factors were found to contribute to the spontaneous loss of infectivity and decay of the virus. These were kinetically and biochemically characterized and now provide a standard which will serve to improve reliability of future anti-viral and vaccine in vitro analysis.